Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane protein. LAT is a critical transmembrane adaptor protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these other adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, genetic and microscopic techniques are currently employed to study the characteristics of LAT-based signaling complexes.A critical pathway activated after TCR engagement and primarily dependent on the LAT molecule is the ERK enzymatic pathway. A number of our older studies demonstrated how interaction of the enzyme phospholipase C gamma (PLCgamma) with a particular phosphorylated tyrosine of LAT results in activation of an enzyme cascade leading to ERK activation. In this pathway PLCgamma activation leads to breakdown of phosphoinositide lipids leading to diacylglycerol production. This product has a number of targets, one of which is the enzyme RasGRP, which activates the small G protein Ras and thereby controls a cascade leading to ERK activation. This route to ERK activation is not the only pathway coupled to LAT leading to ERK activation. The adapter molecule Grb2 also binds phosphorylated LAT and brings the Ras activator molecule SOS1 to LAT as well. In many cell types SOS1 is the central Ras activator. The significance of SOS1 in T cells has been poorly studied. In the previous year to address SOS function we successfully generated a mouse in which SOS1 can be deleted in T cells. As described last year we conducted a series of experiments designed to test the role of SOS1 in thymic development and peripheral T cell function. Our published study indicated that SOS1 is required for the normal development and expansion of immature T cells during intrathymic development (the double negative to double positive phase). At a later stage in intrathymic development during the period when appropriate TCRs are selected (the double positive phase), SOS1 seemed less important in the previous study. We also found that SOS1 is most abundant in the earliest of thymocytes (the double negative phase) and it decreases over the course of thymocyte maturation. This year we continued our work on Ras activation by studying the relative importance of RasGRP and two SOS isoform, SOS1 and SOS2, in thymocyte development and signaling using combinations of genetic models in which these enzymes were deleted. The primary function of SOS1 at the first stage of intrathymic development was confirmed, but an additional role for RasGRP at this stage was demonstrated. The later stage of thymic selection is the phase when positive and negative selection of thymocytes occurs. Positive selection leads to expansion of those thymocytes that mature into T cells that migrate out of the thymus. We confirmed that RasGRP is needed for positive selection. Negative selection is a process that results in the deletion of self-reactive thymocytes. We showed for the first time that deletion of either RasGRP or SOS1 had no effect on this process, but surprisingly, the loss of both of these enzymes disrupted negative selection. These published studies of the last two years together provide important information for understanding the central role of Ras and Ras activators in thymocyte development.In addition to biochemical and genetic studies of signaling molecules the laboratory has developed new methods of visualizing T cell activation using confocal microscopy. Many of the signaling molecules involved in the early TCR-coupled activation process have been tagged with fluorescent markers and expressed in T cells. The group has used these methods to observe the process of the assembly of signaling molecules into signaling clusters at the site of T cell activation using confocal microscopy. This year we completed an extensive set of experiments using a new microscopic technique. High-resolution microscopy is a means to visualize intracellular elements at the level of single molecule resolution, a level of resolution below the limits of diffraction inherent in standard light microscopy. We used one and two color photo-activated localization microscopy, a type of high-resolution microscopy, to study signaling molecules and their organization before and after T cell activation. The interaction of the TCR, ZAP-70 and LAT changed upon activation and interaction of the activated TCR with LAT was highly localized. LAT was found in small nanoclusters before activation and the size distribution of these clusters increased slightly upon activation of the T cell. These LAT nanoclusters had an unexpected structure, as the adapter molecule SLP-76, which is linked to actin polymerization, was found at the periphery of LAT nanoclusters. Much additional work is currently underway on the organization of these and other signaling molecules both in resting and activated T cells.